DNA sequencing, i.e., determining the sequence or order of the nucleotides or bases comprising the DNA, is one of the cornerstone analytical techniques of modern molecular biology. The development of reliable methods of sequencing has led to great advances in the understanding of the organization of genetic information and has made possible the manipulation of genetic material, i.e., genetic engineering.
These are currently two general methods for sequencing DNA: the Maxam-Gilbert chemical degradation method [A. M. Maxam et al., Meth. in Enzym. 65 499-599 (1980)] and the Sanger dideoxy chain terminatin method (F. Sanger, et al., Proc. Nat. Acad. Sci. USA 74 5463-5467 (1977)]. A common feature of these two techniques is the generation of four groups of labeled DNA fragments, each group having a family of labeled DNA fragments with each family containing fragments having differing numbers of nucleotides. The population of fragments within these groups all end with one of the four nucleotides or bases comprising the DNA. Both techniques also utilize a radioactive isotope, such as .sup.32 P or .sup.35 S, as the means for labeling the fragments. The primary difference between the techniques is in the way the fragments are prepared.
In both methods, base sequence information which generally cannot be directly determined by physical methods must be converted into chain-length information which can be determined. This determination can be accomplished through electrophoretic separation. Under denaturing conditions (high temperature, urea present, etc.) short DNA fragments migrate through the electrophoresis medium as stiff rods. If a gel is employed for the electrophoresis, the DNA fragments will be sorted by size and result in a DNA sequence determination with single-base resolution up to several hundred bases.
The Sanger and Maxam-Gilbert methods for DNA sequencing are conceptually elegant and efficacious but they are operationally difficult, time-consuming, and often inaccurate. Many of the problems stem from the fact that the single radioisotopic reporter cannot distinguish between bases. The use of a single reporter to analyze the sequence of four bases lends considerable complexity to the overall process. To determine a full sequence, the four sets of fragments produced by either Maxam-Gilbert or Sanger methodology are subjected to electrophoresis in four parallel lanes. This results in the fragments being spacially resolved along the length of the gel according to their size. The pattern of labeled fragments is typically read by autoradiography which shows a continum of bands distributed between four lanes often referred to as a sequencing ladder. The ladder is read by visually observing the film and determining the lane in which the next band occurs for each step on the ladder. Thermally induced distortions in base mobility in the gel (this usually appears as a "smile effect" across the gel) can lead to difficulties in comparing the four lanes. These distortions often limit the number of bases that can be read on a single gel.
Problems relating to use of a single radioisotopic reporter revolve around its lack of sensitivity and the time required to evaluate a sample. The long times required for autoradiographic imaging aling with the necessity of using four parallel lanes force one into a "snapshot" mode of visualization. Since one needs simultaneous spatial resolution of a large number of bands one is forced to use large gels. This results in additional problems. Large gels are difficult to handle and are slow to run, adding even more time to the overall process.
Once the exposed image of the gel pattern is obtained, there is a problem of visual interpretation. Conversion of a sequencing ladder into a base sequence is a time-intensive, error prone process requiring the full attention of a highly skilled operator. Some mechanical aids do exist but the process of interpreting a sequence gel is still painstaking and slow. Finally, the use of radioactive materials has health risks associated with continued exposure over extended periods. Appropriate use of shielding and disposal procedures imposes some control of exposure levels, but elimination of isotope use would be highly desirable.
To solve these problems, efforts are underway to replace autoradiography with some alternate, non-radiosotopic reporter/detection system using fluorescence. DNA frequencies labeled with one or more fluorescent tags (fluorescent dyes) and excited with an appropriate light source give characteristic emissions from the tags which identify the fragments.
The use of a fluorescent tag as opposed to a radioisotopic level allows one to specify a DNA fragment detection system that responds to the optical parameters characterizing tag fluorescence. For example, the use of four different fluorescent tags, distinguishable on the basis of some emission characteristic (e.g., spectral distribution, life-time, polarization), allows one to uniquely link a given tag with the sequencing fragments associated with a given base. Once such a linkage is established, one can then combine and resolve the fragments from a single sample and make the base assignment directly on the basis of the chosen emission characteristic. When electrophoresis is chosen as a separation means, for example, a single sample containing DNA fragments with base-specific fluorescent tags can be separated in a single gel lane.
The "real-time" nature of fluorescence detection allows one either to scan in the electrophoresis direction a gel containing spatially resolved bands (resolution in space) or to monitor at a single point on the gel and detect bands of separated fragments as they pass in sequence through the detection zone (resolution in time). Large gels are not necessarily required to discriminate between the fragments when time resolution is selected. Furthermore, a "real-time", single lane detection mode is very amenable to fully automated base assignment and data transfer.
A known "real time" fluoresence-based DNA sequencing system developed by the California Institute of Technology is disclosed in at least one published patent application and at least two journal articles: L. M. Smith, West German Pat. Appl. DE No. 3446635 Al (1985); L. M. Smith et al., Nucleic Acids Research, 13 2399-2412 (1985) and L. M. Smith et al., Nature, 321: 674-679 (1986). This system employs four sets of DNA sequencing fragments, each labeled with one of four specified fluorescent dyes. Unfortunately, the fluorescence (emission) maxima are spread over a large wavelength range (approximately 100 nm) to facilitate discrimination among the four dyes, but, the absoption (excitation) maxima for the dyes are comparably spread. This makes it difficult to efficiently excite all four dyes with a single monochromatic source and adequately detect the resulting emissions.
It would be preferable to use dyes with closely spaced absorption (and corresponding emission) spectra, selected to enhance the excitation efficiency. But such closely spaced spectra cause other difficulties. Recalls that a real time detection system for DNA sequencing must be able to distinguish between four different dye emission spectra in order to identify the individual labeled fragments. The emissions are typically of relatively low intensity. The detection system must have a high degree of selectivity and sensitivity (better than 10 (-16) moles DNA per band), and a means to minimize stray light and background noise, in order to meet desired performance characteristics. The system must also be able to monitor the detection area frequently enough to avoid missing any fragments that may migrate past the detection window between scans.
In order to effectively utilize an electrophoresis gel, a typical DNA sequencing experiment involves running multiple samples simultaneously in parallel lanes of a slab gel. Therefore, an excitation/detection system must also be able to monitor each lane of such a gel at essentially the same time. A system must be capable of monitoring a detection zone which spans the majority of the usable gel width. Typical sequencing gels have lanes that are 4-5 mm wide with 1-2 mm spacing between lanes. Therefore, in order to monitor a 10 lane gel, a detection system must excite and detect emissions from a region typically as wide as 70 mm.
Another fluorescence detection system developed for similar applications, is disclosed in a U.S. patent application Ser. No. 07/057,566 filed June 12, 1987 by Prober et al. This application discloses a system for detecting the presence of fluorescent energy from different species, typically dye-labeled DNA, following separation in time and/or space, and identifying the species. A set of four labels are chosen such that all four are efficently excited by a single source, yet have emission spectra that are similar but distinguishable in wavelength. Since differential perturbations in electrophoretic mobility of the attached DNA fragments are small, any disturbance to this behavior is minimized by using four tags that have similar molecular weights, shape and charge.
The scheme of Prober et al. provides for modulating and ratioing the signals corresponding to the fluorescent energies in two different wavelength ranges to obtain a resultant signal that determines the identity of the species. A dichoroic filter, with a transmission/reflection characteristic that various as a function of wavelength, or two filters with passbands that vary as a function of wavelength, effect the modulation. Two detectors are positioned respectively to receive the transmitted and reflected emissions and generate first and second signals that vary in different senses corresponding to the intensities of each. Preferably the dichroic filter characteristic has a relatively sharp transition from transmission to reflection which occurs near the center of the species emission spectra.
This system overcomes many of the problems of Smith et al. and has the ability to distinguish in real time between relatively small wavelength differences in emission spectra, while maintaining a relatively high degree of sensitivity. Further, the system delivers a high portion of the usable light onto the two photometric detectors to maintain continuous monitoring of the gel containing the fluorescent species.
Both of the systems described above operate a fixed light beam and fixed detectors which together can monitor only a single point within the monitoring region. In order to monitor more than one spacial position (lane or lane position) of a gel, either the light beam must be scanned while providing a means to detect the emissions from the dyes, or the gel must by physically shifted while holding the beam fixed. The latter of the two alternatives, moving the gel, is not always practical since a large electrophoresis gel along with its associated buffer reservoirs are physically cumbersome. The other alternative, moving the beam while the gel is stationary, has its own problems since the detectors must remain closely coupled to sources of emission to prevent the entry of stray light and maximize collection of the emitted light.
One method of accomplishing this task is to physically move either of the two previously discussed detection systems and their associated optics and light beam so that several lanes in the gel are effectively scanned. This type of system has the disadvantage of being mechanically complex while introducing additional noise into the system. Reliability and the high costs associated with this type of system would also be a concern.
Another known "scanning" detection system is discussed in U.S. Pat. No. 3,764,512 issued to Green et al. This system discloses a laser scanning electrophoresis instrument and system for determining the electrokinetic or zeta potential of dispersed particles in an aqueous solution. This system utilizes a galvanometer mirror to scan a laser light beam across an electrophoresis medium. The system is not capable of detecting multiple samples moving perpendicular to the scanning motion of the beam.
Another scanning system is disclosed in U.S. Pat. No. 4,162,405, Chance et al. which describes an apparatus for measuring the heterogeneity of oxygen delivery to perfused and in situ organs. A laser is employed as a flying spot scanning excitation source and uses two photodetectors to monitor the emission signal and excitation wavelength light. Although an x-y scanner is used to move the laser beam over the sample area, the total scanned area is only 1 cm by 1 cm. (As mentioned earlier for a multiple sample DNA sequencer, a sample area 7 times wider is needed).
To implement the schemes of Chance et al. for a large area, the detectors must be either larger in size, or located further from the sample thus diminishing the collection efficiency. Furthermore, the detection of closely spaced emission spectra of relatively low light intensities in the presence of a much more intense excitation source requires the selective transmission properties offered by interference filters. In order to monitor a relatively large spacial area, both large detectors and large filters must be used. Unfortunately, large interference filters that collect light even a large solid angle are subject to transmission properties which vary with the angle of incidence of the light. Thus, when placed close to the emission source, light impinging on the filter with an angle of incidence greater than about 22 degrees can experience significantly less rejection of the excitation light than light at normal incidence. Consequently, if the filter subtends a relatively large solid angle with respect to the source of emission, the overall excitation wavelength rejection properties of the filter will be compromised due to leakage of excitation light entering at the higher angles of incidence.